Peptides reversed-phase liquid chromatography

Figure 9.10 Three-dimensional representation of the data volume of a tryptic digest of ovalbumin. Series of planar slices through the data volume produce stacks of disks in order to show peaks. Reprinted from Analytical Chemistry, 67, A. W. Moore Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatogra-phy/reversed phase liquid chromatography/optically gated capillary zone electrophoresis, pp. 3456-3463, copyright 1995, with permission from the American Chemical Society.

A. W. Moore, Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/ optically gated capillary zone electrophoresis . Anal. Chem. 67 3456-3463 (1995). 

Holland, L.A., Jorgenson, J.W. (2000). Characterization of a comprehensive two-dimensional anion exchange-perfusive reversed phase liquid chromatography system for improved separations of peptides. J. Microcolumn. Sep. 12, 371-377. 

Banks, J. F., Gulcicek, E. E. (1997). Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with online electrospray time of flight mass spectrometry. Anal. Chem. 69(19), 3973-3978. 

Chen, Y., Mehok, A.R., Mant, C.T., Hodges, R.S. (2004). Optimum concentration of trifluor-oacetic acid for reversed-phase liquid chromatography of peptides revisited. J. Chromatogr. A 1043, 9-18. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

A. General description Eptifibatide is a cyclic heptapeptide containing six amino acids and one mercaptopropionyl residue. An interchain disulfide bridge is formed between the cysteine amide and the mercaptopropionyl moieties. Eptifibatide binds to the platelet receptor glycoprotein (gp) Ilb/IIIa of human platelets and inhibits platelet aggregation. The eptifibatide peptide is produced by solution-phase peptide synthesis, and is purified by preparative reverse-phase liquid chromatography and lyophifized. 

The use of reversed-phase liquid chromatography is growing in applications for separating mixtures of peptides. In this type of chromatography, the stationary phase is nonpolar, whereas the mobile phase is polar. The stationary phase is normally porous silica with bonded n-alkyl chains, mainly octadecyl but also octyl, hexyl, butyl, and propyl chains. 

Fujinary E, Manes JD, Bizanek R, Peptide content determination of crude synthetic peptides by reversed-phase liquid chromatography and nitrogen-specific detection with a chemiluminescent nitrogen detector, J. Chromatogr A, 743 85-89, 1996. 

This work uncovered the fact that a substitution of the amino acid alanine for valine at position 126 in the /3-chain of hemoglobin occurred in a hemato-logically normal adult of Lebanese extraction. The variation /3-globin was initially observed and subsequently purified by reverse-phase liquid chromatography (see Fig. 2-5). Also LC was used to isolate the variant tryptic peptide of /3-T13 that had alanine replacing the valine at amino-acid residue position 126. This is shown in Figure 2-6. (Peptide mapping is discussed later in this chapter.)... 

Competitive ionization may be avoided by varying the pH conditions or the matrix, through chemical derivatization of the peptides contained in this mixture, or through the partial fractionation of the mixture through reversed-phase liquid chromatography so that each fraction contains peptides of a similar hydrophobicity. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

Petritis, K. et al. Ion-pair reversed-phase liquid chromatography-electrospray mass spectrometry for the analysis of underivatized small peptides. /. Chmmatogr. A. 2002, 957,173-185. 

Wang, X. and Carr, P.W. Unexpected observation concerning the effect of anionic additives on the retention behavior of basic drugs and peptides in reversed-phase liquid chromatography. J. Chromatog. A. 2007, 1154, 165-173. 

The ion-exchange constant does vary from one pair of ions to another therefore selectivity can be controlled by appropriate choice of the counter ion. However, selectivity is more conveniently manipulated by controlling the pH of the mobile phase and taking advantage of differences in the values of the analytes to be separated. In contrast with reversed-phase liquid chromatography (see section 3.6.2.1) the retention of weak acids and weak bases will reach a maximum when the compounds are in their ionized forms. Zwitterionic compounds such as amino acids, peptides and proteins can be separated on anion exchangers or cation exchangers. 

Figure 4 Depiction of shot-gun proteomics using multidimensional protein identification technology a complex mixture of peptide fragments in the digest are resolved by a combination of ion-exchange and reversed-phase liquid chromatography.

Figure 10.9 shows an example of peptide separations obtained with reversed-phase liquid chromatography. Tte sample is the tryptic digest of bovine... 

If the tissue component is very complex, or if the peptide of interest is only a minor component of the sample, a fractionation by means of (micro-) reversed-phase liquid chromatography should be earned out The collected fractions may then be assayed by mass spectrometry. 

Lee, Y.H. Kim, M.-S. Choie, W.-S. Min, H.-K. Lee, S.-W. Highly informative pro-teome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry. Prvteomics 2004,4,1684-1694. 

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